JMJD3 Is Required for Acute Pancreatitis and Pancreatitis-Associated Lung Injury.

Acute pancreatitis (AP) can be complicated by inflammatory disorders of remote organs, such as lung injury, in which Jumonji domain-containing protein 3 (JMJD3) plays a vital role in proinflammatory responses. Currently, we found that JMJD3 expression was upregulated in the pancreas and lung in an AP male mouse model, which was also confirmed in AP patients. Further experiments revealed that the upregulation of JMJD3 and proinflammatory effects were possibly exerted by mitochondrial DNA (mtDNA) or oxidized-mtDNA from tissue injury caused by AP. The release of mtDNA and oxidized-mtDNA contributed to the infiltration of inflammatory monocytes in lung injury through the stimulator of IFN genes (STING)/TLR9-NF-κB-JMJD3-TNF-α pathway. The inhibition of JMJD3 or utilization of Jmjd3-cKO mice significantly alleviated pulmonary inflammation induced by AP. Blocking mtDNA oxidation or knocking down the TLR9/STING pathway effectively alleviated inflammation. Therefore, inhibition of JMJD3 or STING/TLR9 pathway blockage might be a potential therapeutic strategy to treat AP and the associated lung injury.

Generation of Highly Biomimetic Quality Control Materials for Noninvasive Prenatal Testing Based on Enzymatic Digestion of Matched Mother-Child Cell Lines.

BACKGROUND: Noninvasive prenatal testing (NIPT) based on cell-free DNA (cfDNA) is widely used. However, biomimetic quality control materials that have properties identical to clinical samples and that are applicable to a wide range of methodologies are still not available to support assay development, internal quality control, and proficiency testing. METHODS: We developed a set of dual enzyme-digested NIPT quality control materials (DENQCMs) that comprise simulated human plasma and mixtures of mother cell line-derived cfDNA based on DNA fragmentation factor digestion (D-cfDNA) and the matched child cell line-derived cfDNA based on micrococcal nuclease digestion (M-cfDNA). Serially diluted samples positive for trisomies 21, 18, and 13 were included in the materials. To evaluate the biomimetics, DENQCMs were analyzed using random massively parallel sequencing (MPS), targeted MPS, and imaging single DNA molecule methods, and the estimated fetal fractions (FFs) were compared with expected FFs. Genome-wide analysis of cfDNA fragmentation patterns was performed to confirm their biological characteristics. RESULTS: The genetic status of each DENQCM was correctly detected by 4 routine NIPT assays for the samples with FFs >5%. The chromosome Y-based and single-nucleotide polymorphism-based estimations of FFs were linearly related to those expected FFs. The MPS results exhibited a concordance of quality metrics between DENQCMs and maternal plasma, such as GC contents of cfDNA and unique read ratios. CONCLUSIONS: The DENQCMs are universally applicable for different platforms. We propose DENQCMs as an approach to produce matched maternal and fetal cfDNA that will be suitable for the preparation of quality control materials for NIPT.

A novel cell line generated using the CRISPR/Cas9 technology as universal quality control material for KRAS G12V mutation testing.

BACKGROUND: KRAS mutations are the key indicator for EGFR monoclonal antibody-targeted therapy and acquired drug resistance, and their accurate detection is critical to the clinical decision-making of colorectal cancer. However, no proper quality control material is available for the current detection methods, particularly next-generation sequencing (NGS). The ideal quality control material for NGS needs to provide both the tumor mutation gene and the matched background genomic DNA, which is uncataloged in public databases, to accurately distinguish germline polymorphisms and somatic mutations. METHODS: We developed a novel KRAS G12V mutant cell line using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technique to make up for the deficiencies in existing quality control material and further validated the feasibility of the cell line as quality control material by amplification refractory mutation system (ARMS), Sanger sequencing, digital PCR (dPCR), and NGS. RESULTS: We verified that the edited cell line specifically had the G12V mutation, and the validation results presented a high consistency among the four methods of detection. The three cell lines screened contained the G12V mutation and the mutation allele fractions of G12V-1, G12V-2, and G12V-3 were 52.01%, 82.06%, and 17.29%, respectively. CONCLUSION: The novel KRAS G12V cell line generated using the CRISPR/Cas9 gene editing system is suitable as a quality control material for all current detection methods and provides a new direction in the development of quality control material.

Synthetic Circulating Cell-free DNA as Quality Control Materials for Somatic Mutation Detection in Liquid Biopsy for Cancer.

BACKGROUND: Detection of somatic genomic alterations in tumor-derived cell-free DNA (cfDNA) in the plasma is challenging owing to the low concentrations of cfDNA, variable detection methods, and complex workflows. Moreover, no proper quality control materials are available currently. METHODS: We developed a set of synthetic cfDNA quality control materials (SCQCMs) containing spike-in cfDNA on the basis of micrococcal nuclease digestion carrying somatic mutations as simulated cfDNA and matched genomic DNA as genetic background to emulate paired tumor-normal samples in real clinical tests. Site-directed mutagenesis DNA that contained 1500-2000 bases with single-nucleotide variants or indels and genomic DNA from CRISPR/Cas9 edited cells with EML4-ALK rearrangements was fragmented, quantified, and added into micrococcal nuclease-digested DNA derived from HEK293T cells. To prove their suitability, the SCQCMs were compared with patient-derived plasma samples and validated in a collaborative study that encompassed 11 laboratories. RESULTS: The results of SCQCM analysis by next-generation sequencing showed strong agreement with those of patient-derived plasma samples, including the size profile of cfDNA and the quality control metrics of the sequencing data. More than 95% of laboratories correctly detected the SCQCMs with EGFR T790M, L858R, KRAS G12D, and a deletion in exon 19, as well as with EML4-ALK variant 2. CONCLUSIONS: The SCQCMs were successfully applied in a broad range of settings, methodologies, and informatics techniques. We conclude that SCQCMs can be used as optimal quality controls in test performance assessments for circulating tumor DNA somatic mutation detection.

Assessment of significant procedures in multigene molecular detection for breast cancer in clinical laboratories: from variant detection to targeted therapy.

BACKGROUND: In recent years, numerous novel targeted drugs against breast cancer have been developed because of the rapid progress in multigene molecular testing based on next-generation sequencing (NGS). However, it is a great challenge for clinicians to update the drug information timely, therefore necessitating that clinical laboratories provide adequate and comprehensive targeted drugs information to clinicians as a reference. The premise of providing this information is the accuracy of variant detection. Our study aimed to assess the entire process of variant detection, interpretation, and targeted therapy. METHODS: Laboratories were instructed to use routine methods for variant detection. The results were evaluated based on a predefined score system, and differences in variant interpretation were analyzed. Targeted drug information provided by laboratories was also summarized, and its accuracy and sufficiency were assessed. RESULTS: Overall, 90.1% (82/91) of the laboratories produced accurate results. 78.9% (15/19) of the errors were false positives or false negatives. Incorrect and insufficient drug information was mainly provided due to failure in timely database updating, inconsistencies with the detected mutations or given clinical information, and negligence during phase I clinical trials. To prioritize providing targeted drug information, laboratories collected data were based on different factors, including variant clinical significance, allele frequency, and variant positions in the signal pathway. CONCLUSION: The variant detection capability was satisfactory, but the ability to provide accuracy and comprehensive targeted drug information should be urgently improved. Our study summarized a completed NGS-based multigene molecular detection pipeline, aiming to better inform precision treatment for breast cancer patients.

Challenges of Providing Concordant Interpretation of Somatic Variants in Non-Small Cell Lung Cancer: A Multicenter Study.

Background: Success of multiple-gene mutation tests by next-generation sequencing (NGS), associated with molecular targeting therapies for cancers, depending on the accuracy and consistency of interpreting variants. Here, we summarized reports from clinical laboratories for cases with non-small cell lung cancer (NSCLC) and discussed conflicting interpretations of somatic variants. Methods: Three mimetic DNA samples, containing six somatic mutations, were prepared based on three clinical case reports of NSCLC. Clinical reports and genetic testing questionnaires were collected from 67 laboratories enrolled in this investigation. Results: Thirty-four laboratories with correct variant results identified two variants, based on FDA approval of targeted drugs for the same tumor, consistently, with strong clinical significance, whereas the other variants were classified with conflicting interpretations. Discordant interpretations were reported for ERBB2 with three different classifications, including strong clinical significance (53.0%, 18/34), potential clinical significance (38.2%, 13/34), and unknown significance (8.8%, 3/34). In the variant therapeutic drug recommendation section, 32.4% of the laboratories (11/34) did not recommend all the available therapeutic drugs designated by the National Comprehensive Cancer Network (NCCN). In the remaining group of 33 laboratories with incorrect variant results, less correct classifications were acquired for the variants with strong clinical significance. Conclusions: Owing to numerous reasons, the interpretation of variants differed greatly, which might in turn lead to the inappropriate clinical care of patients with NSCLC. By analyzing the limitations of different databases used by laboratories, we integrated various types of databases with different levels of evidence to form a comprehensive and detailed variant interpretation pipeline, aiming to standardize the variant classification and provide accurate and sufficient therapeutic drug recommendation to clinicians for minimal-inappropriate therapeutic options.

Prognostic value of EGFR and KRAS in circulating tumor DNA in patients with advanced non-small cell lung cancer: a systematic review and meta-analysis.

EGFR (exon 19 and exon 21) mutations in patients with advanced non-small cell lung cancer (NSCLC) treated by EGFR-TKIs are associated with a better survival; while KRAS mutations predict a worse prognosis. However, there are divergent findings regarding the prognostic value of EGFR and KRAS mutations in circulating tumor DNA (ctDNA). We aimed to summarize the evidence for the use of circulating EGFR and KRAS mutations as prognostic factors in advanced NSCLC patients.We searched the network databases for studies reporting progression-free survival (PFS) and overall survival (OS) stratified by EGFR or KRAS mutations in ctDNA in advanced NSCLC patients. Thirteen studies enrolling 2,293 patients were reviewed. Correlation of circulating EGFR or KRAS mutations with patients' prognosis was assessed by meta-analysis.The pooled analyses showed that EGFR mutations in ctDNA significantly prolong PFS (HR=0.64,95% CI 0.51-0.81, I2=0%, p=0.0002), namely, in patients treated by EGFR-TKIs. There is a trend to have a prolonged OS for advanced NSCLC patients with circulating EGFR mutations who were treated by EGFR-TKIs (HR=0.79, 95% CI 0.52-1.21, I2=0, p=0.28). KRAS mutations detected in ctDNA predict a worse PFS (HR=1.83, 95% CI 1.40-2.40, p<0.0001) and OS (HR=2.07, 95% CI 1.54-2.78, p<0.00001) in advanced NSCLC patients treated by chemotherapy. Sensitivity analyses and subgroup analyses demonstrated the stability of our conclusion.Our analysis showed that EGFR mutations in ctDNA predicted a better PFS, in particular in advanced NSCLC patients treated by EGFR-TKIs. KRAS mutations in ctDNA indicated a worse PFS and OS in patients treated by chemotherapy.

Prognostic value of circulating tumor DNA in patients with colon cancer: Systematic review.

The application of circulating tumor DNA(ctDNA) represents a non-invasive method for tumor detection. Its prognostic significance in patients with colorectal cancer is controversial. We performed a systematic review of data from published studies to assess the prognostic values of ctDNA in patients with colorectal cancer. We searched Medline, Embase, Web of Science, the Cochrane Library, and Scopus databases to identify eligible studies reporting disease-free survival (DFS) and overall survival (OS) stratified by ctDNA prior to December 6, 2016. We evaluated the quality and design of these studies. A total of 22 studies were eligible for systematic review. Among them, 11 studies investigated the prognostic value of ctDNA on disease-free survival (DFS). Seven of 11 studies showed that ctDNA was an independent variable to estimate the probability of DFS by multivariate analyses. Thirteen studies assessed the relationship between ctDNA and overall survival (OS). Eight of 13 studies showed that ctDNA was an independent predictor of worse OS through the use of multivariate analyses. This analysis provides evidence that ctDNA may be a prognostic biomarker, negatively correlated with the survival of patients with colorectal cancer.

[Emergence of methicillin-resistant Staphylococcus aureus SCCmec type IV/V epidemic clones in a large teaching hospital in China].

OBJECTIVE: To investigate the staphylococcal cassette chromosome mec (SCCmec) genotype and molecular epidemiological characteristics of healthcare-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) in a large teaching hospital in China. METHDOS: From January 2012 to December 2012, a total of 71 nonduplicate HA-MRSA were collected in a teaching hospital in Changsha, China. SCCmec types were determined by multiplex PCR, and Panton-Valentine leukocidin (PVL) gene was detected by PCR. The homology among the tested isolates was determined using pulsed-field gel electrophoresis (PFGE). RESULTS: Of the 71 HA-MRSA isolates, 49 (69.0%) carried SCCmec III, 10 (14.1%) carried SCCmec IV, 3 (4.2%) carried SCCmec V and 3 (4.2%) carried SCCmec II; the remaining 6 isolates were not typeable by PCR. Compared with patients having SCCmec I/II/III MRSA infections, those with SCCmec IV/V MRSA infections had a significantly younger age and a similar duration of hospital stay before the first MRSA-positive culture and total hospital stay. PVL genes were strongly associated with SCCmec type IV/V MRSA infections. HA-SCCmec IV/V MRSA strains showed a greater susceptibility to rifampicin, gentamicin, levofloxacin, ciprofloxacin, and tetracycline than HA-SCCmec I/II/III MRSA strains. The 13 HA-SCCmec IV/V MRSA isolates formed one large group at the 55% similarity level. Three PFGE clusters with a similarity index of 85% or more were identified, and unique PFGE profiles were observed in 4 isolates. CONCLUSION: This is the first report of HA-MRSA isolates carrying SCCmec V in Chinese hospitals. SCCmec types IV and V MRSA clones have emerged in Chinese hospitals, which urges more rigorous surveillance of their spread in healthcare facilities in China.

Technical Validation of a Next-Generation Sequencing Assay for Detecting Clinically Relevant Levels of Breast Cancer-Related Single-Nucleotide Variants and Copy Number Variants Using Simulated Cell-Free DNA.

Next-generation sequencing (NGS) is commonly used in a clinical setting for diagnostic and prognostic testing of genetic mutations to select optimal targeted therapies. Herein, we describe the development of a custom NGS assay for detecting single-nucleotide variants (SNVs) and copy number variations (CNVs) in a panel of 51 genes related to breast cancer. We designed and implemented a validation strategy in accordance with principles and guidelines developed by the Next-Generation Sequencing: Standardization of Clinical Testing work group using artificial, cell-free DNA (cfDNA) with mutant fragments prepared in a simple, rapid, and cost-effective manner. For SNV detection, our test had 96.30% sensitivity at mutant allele frequency ≥0.5% with high specificity (99.9997%) and accuracy (99.9996%). For CNV detection, the approach had 95.83% sensitivity for copy numbers at 1.25× (25.6% extra copies) with high specificity (99.77%) and accuracy (99.76%). In addition, our NGS-based assay demonstrated high intrarun and interrun reproducibility, high consistency compared to digital PCR, and a low cross-contamination rate. An overall assessment using cfDNA and plasma cfDNA samples demonstrated our custom NGS assay yields a reliable and robust detection sensitivity with a mutant allele frequency as low as 0.5% for SNVs and copy number of 1.25× for CNVs.

Reliability Assurance of Detection of EML4-ALK Rearrangement in Non-Small Cell Lung Cancer: The Results of Proficiency Testing in China.

INTRODUCTION: Currently, several approaches are being used to detect echinoderm microtubule associated protein like 4 gene (EML4)-anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangement, but the performance of laboratories in China is unknown. To evaluate the proficiency of different laboratories in detecting EML4-ALK rearrangement, we organized a proficiency test (PT). METHODS: We prepared formalin-fixed, paraffin-embedded samples derived from the xenograft tumor tissue of three non-small cell lung cancer cell lines with different EML4-ALK rearrangements and used PTs to evaluate the detection performance of laboratories in China. RESULTS: We received results from 94 laboratories that used different methods. Of the participants, 75.53% correctly identified all samples in the PT panel. Among the errors made by participants, false-negative errors were likely to occur. According to the methodology applied, 82.86%, 76.67%, 77.78%, and 66.67% of laboratories using reverse transcriptase polymerase chain reaction, fluorescence in situ hybridization, next-generation sequencing, and immunohistochemical analysis, respectively, could analyze all the samples correctly. Moreover, we have found that the laboratories' genotyping capacity is high, especially for variant 3. CONCLUSION: Our PT survey revealed that the performance and methodological problems of laboratories must be addressed to further increase the reproducibility and accuracy of detection of EML4-ALK rearrangement to ensure reliable results for selection of appropriate patients.

Comparison of the types of candidate reference samples for quality control of human epidermal growth factor receptor 2 status detection.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) is as a target gene for trastuzumab in patients with breast cancer. Accurate determination of HER2 status and strict quality control are necessary to ensure reproducibility and accuracy of the techniques used for the determination of HER2 status. METHODS: We used three different types of samples: formalin-fixed and paraffin-embedded (FFPE) samples prepared from cell lines, agarose gel samples using cell lines, and xenograft tumor samples. One cell line for FFPE or xenografts did not overexpress HER2, while the others showed different levels of HER2 overexpression. We compared the morphology, HER2 gene amplification status, and HER2 protein expression status of these samples with those of clinical specimens. RESULTS: We successfully produced three kinds of samples for quality control. Cells from the cell line-sample sections were dispersed while those from the agarose gel-sample sections and xenograft tumor sample sections (prepared from the both cell lines) were concentrated in one area. The FISH results for all three kinds of samples were as expected. The IHC results of the cell line samples and xenograft tumor samples were as expected, but the staining level of the agarose gel samples, using HER2-overexpressed cell lines was weak which might be regarded as a false negative result. CONCLUSIONS: Xenograft tumor samples might be used as an additional option for quality control in FISH and IHC. However, it might not replace the clinical specimen quality controls directly.

The reliable assurance of detecting somatic mutations in cancer-related genes by next-generation sequencing: the results of external quality assessment in China.

To evaluate the proficiencies of laboratories utilizing next-generation sequencing (NGS) to detect somatic mutations in cancer-related genes, an external quality assessment (EQA) was implemented by the National Center for Clinical Laboratories of China in 2015. We prepared a panel of samples that comprised eight samples made by mixing synthetic mutated DNA fragments with normal human genomic DNA and one reference sample containing only genomic DNA. We validated our sample panel, and then distributed it to laboratories across China. We received complete results from 64 laboratories. The performances of 51.6 % (33/64) respondent labs were acceptable and 26.6 % (17/64) of the labs returned perfect results. In total, 449 mistakes were reported, including 201 false-negatives (201/449, 44.8 %) and 222 false-positives (222/449, 49.4 %) and 26 slightly discordant results (26/449, 5.8 %). We believe these unsatisfactory results and varied performances are mainly due to the enrichment methods used, the diverse sequencing chemistries of the different NGS platforms, and other errors within the sequencing process. The results indicate that our sample panel is suitable for use in EQA studies, and that further laboratory training in targeted NGS testing is urgently required. To address this, we propose a targeted NGS workflow with details on quality assurance procedures according to the current guidelines.

Hybrid kappa\lambda antibody is a new serological marker to diagnose autoimmune pancreatitis and differentiate it from pancreatic cancer.

The only generally accepted serological marker currently used for the diagnosis of autoimmune pancreatitis (AIP) is IgG4. Our aim was mainly to determine whether hybrid κ\λ antibody can help to diagnose AIP and to differentiate it from pancreatic cancer. We established an enzyme-linked immunosorbent assay (ELISA) system to measure the levels of hybrid κ\λ antibodies in human sera. Sera were obtained from 338 patients, including 61 with AIP, 74 with pancreatic cancer, 50 with acute pancreatitis, 40 with ordinary chronic pancreatitis, 15 with miscellaneous pancreatic diseases, and 98 with normal pancreas. Our study showed levels of hybrid κ\λ antibodies in the AIP group were significantly higher than in the non-AIP group (P < 0.001). The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the diagnosis of AIP were 80.3%, 91%, 66.2% and 95.5% respectively. Furthermore, the combined measurement of serum hybrid κ\λ antibody and IgG4 tended to increase the sensitivity although the difference was not statistically significant (90.2% vs. 78.7%, P = 0.08), compared to measurement of IgG4 alone. Our findings suggest that hybrid κ\λ antibody could be a new serological marker to diagnose AIP and differentiate it from pancreatic cancer.

The evaluation of 7 commercial real-time PCR kits for Zaire ebolavirus using virus-like particle-encapsulated EBOV RNA.

Several Chinese commercial real-time PCR kits for Zaire ebolavirus have been developed after the Ebola outbreak and used by Chinese medical teams in West Africa. In order to know the essential performance indicators of these kits, analytical sensitivity and precision were evaluated with virus-like particle (VLP)-encapsulated EBOV RNA. The limit of detection (LOD) and the precision were completed with a series of VLPs. The maximum and minimum of LOD was acquired by ZJ BioTech and Daan gene, respectively. For precision, all of the detection results were <5% except the maximum 5.17%. Among them, Puruikang, Daan gene, Sansure, ZJ BioTech, and Huada demonstrated superior reproducibility. Overall, the requirements of LOD <1000 copies/mL and coefficient of variation <5% could be satisfied by all kits except Kehua. Meanwhile, it is feasible for VLPs as a substitute of positive samples in assay evaluation.